MDCK suspension cell lines in serum-free, chemically-defined media for vaccine production
Disclosed is an adapted Madin-Darby canine kidney cell line capable of suspension culture in the absence of serum, and a chemically-defined medium for culture of the adapted MDCK cell line. Further disclosed are culture methods for growing the adapted MDCK cell line and methods for producing a vaccine from the adapted MDCK cell line grown in the chemically-defined medium.
1 . A method of culturing suspension-and serum-free-adapted Madin-Darby canine kidney (MDCK) cells comprising contacting suspension-and serum-free-adapted MDCK cells (i) without microcarriers; and (ii) with chemically-defined and animal-component-free-growth medium, wherein the contacting is between about 1 day and about 12 days without medium exchange, wherein the chemically-defined and animal-component-free-medium comprises about 20 mM to about 30 mM glucose, wherein the culturing is suspension culturing at 5% CO 2 .
2 . The method of claim 1 , wherein the chemically-defined and animal-component-free-growth medium further comprises one or more amino acids selected from L-Serine, L-Arginine HCl, L-Leucine, L-Tyrosine 2Na 2H2O, L-Isoleucine, L-Threonine, L-Valine, L-Cysteine HCl H2O, L-Aspartic Acid, L-Glutamic Acid, L-Asparagine, L-Phenylalanine, L-Histidine HCl H2O, L-Methionine, L-Alanine, or L-Tryptophan.
3 . The method of claim 1 , wherein the suspension-and serum-free-adapted MDCK cells are deposited as DSM ACC3309 with Leibniz-Institut DSMZ-Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH.
4 . The method of claim 1 , wherein the suspension-and serum-free-adapted MDCK cells have an average doubling time of between about 30 hours and about 35 hours.
5 . The method of claim 4 , wherein the suspension-and serum-free-adapted MDCK cells are suitable for producing virus for a human vaccine.
6 . The method of claim 5 , wherein the virus maintains antigenicity.
7 . The method of claim 6 , wherein the virus is an influenza virus.
8 . A method for producing and culturing an isolated suspension of a serum free adapted Madin-Darby canine kidney (MDCK) cell line comprising:
(a) contacting adherent MDCK cells with a growth medium comprising between about 1% v/v to about 20% v/v serum for a period of time sufficient to establish the adherent MDCK cells in culture;
(b) contacting the established adherent MDCK cells with increasing amounts of chemically-defined growth medium and decreasing amounts of the serum serially until the chemically-defined growth medium does not comprise serum and represents all of the medium;
(c) isolating the adapted MDCK cells thereby producing an isolated suspension of a serum free adapted MDCK cell line; and
(d) culturing the isolated suspension of a serum free adapted MDCK cell line between about 1 day and about 12 days with a chemically-defined and animal-component-free-medium without medium exchange and without microcarriers.
9 . The method of claim 8 , wherein the established adherent MDCK cells of (b) are agitated and in the absence of microcarriers.
10 . The method of claim 9 , wherein the isolated suspension of a serum free adapted MDCK cell line is deposited as DSM ACC3309 with Leibniz-Institut DSMZ-Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH.
11 . The method of claim 9 , wherein the growth medium of (a) comprises 5% serum.
12 . The method of claim 11 , wherein the serum is fetal bovine serum (FBS).
13 . The method of claim 12 , wherein the isolated suspension of a serum free adapted MDCK cell line is suitable for producing a virus for a vaccine.
14 . The method of claim 13 , wherein the isolated suspension of a serum free adapted MDCK cell line has a doubling time of between about 30 hours and 35 hours.
15 . The method of claim 14 , wherein the culturing is in suspension at 5% CO 2 .